The search for therapeutics is largely focused on the regulation of gene expression and the inactivation of gene products. The development of in vitro selection techniques for identifying aptamer sequences that specifically bind a desired target molecule has provided new opportunities for the manipulation of biological interactions. Aptamers are nucleic acid molecules that are capable of binding to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers have been successfully produced which bind many different entities from large proteins to small organic molecules. For recent reviews of aptamers and their ligands, see Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9:324-9(1999), and Hermann and Patel, Science 287:820-5 (2000). A variety of approaches have been taken which make use of aptamer sequences in an attempt to alter the behavior and/or populations of biomolecules within cells.
In the most common approach to date, aptamers have been used to disrupt the molecular interactions of the end products of gene expression—proteins. Thus, aptamers have been selected which bind various protein targets and disrupt the interactions of those proteins with other proteins and/or disrupt catalysis by the protein targets. For instance, Blind et al. have shown that an RNA aptamer which specifically binds β2 integrin LFA-1 can shut down a signaling pathway in vivo (Blind et al., Proc. Natl. Acad. Sci. 96:3606-3610 (1999)). As another example, U.S. Pat. No. 5,756,291 discloses DNA aptamers which bind thrombin and inhibit coagulation. For a review of the success of the use of aptamers as therapeutic reagents, see Osborne et al., Curr. Opin. Chem. Biol. 1:5-9 (1997). Such approaches directed towards the protein products of genes, however, are not very efficient in addressing diseases or conditions where an aberrant amount of a protein is expressed and may be useless in the treatment of diseases or conditions where a protein is underexpressed.
In another approach, and one more directed towards modifying gene expression, aptamers have been used to prevent transcription of a gene by specifically binding the DNA-binding sites of regulatory proteins. In this manner, the aptamers effectively compete with the binding sites on the gene for interaction with the regulatory protein. For instance, PCT Publication No. WO 98/29430 teaches modulation of the immune response. In the taught method, aptamers are used to bind the DNA- binding sites of Sp1 and Sp1-related proteins. However, because the binding of the aptamer to the DNA is a competitive process, high levels of active aptamers are required in vivo to achieve reasonable efficiency of gene modulation.
In yet another approach, the PCT Publication No. WO 00/20040 discloses the inhibition of expression of a gene in a cell by contacting a small molecule with an aptamer in the 5′untranslated region (5′UTR) of the gene's mRNA transcript. The binding of the small molecule to the aptamer results in disruption of the translation of the mRNA, leading to a change in gene expression. However, the disclosed technique is limited in that it can only be used to turn off the expression of a gene, not activate it. Furthermore, the degree of downregulation will merely be proportional to the amount of the aptamer's ligand which has been administered to the cell.
Thus, the methods that use aptamers in therapeutics which have been developed to date have not fully addressed the need for methods of efficiently upregulating and downregulating gene expression in a cell in a dose-responsive manner.